Whereas UDP-glucuronosyltransferase (UGT) isozymes detoxify numerous lipophilic-behaving endogenous agents and exogenous chemical toxicants present in our daily diet and environment by linking the same to glucuronic acid, the special properties and enzymatic mechanism(s) that enable endoplasmic reticulum (ER)-bound UGTs to convert structurally diverse lipophiles, including therapeutics, to excretable glucuronides are unknown. We have accumulated evidence that regulated phoshorylation of UGTs is required for their activity and evidently their substrate selections. Inhibition of recombinant UGTs expressed in epithelial cells with various kinase inhibitors, incorporation of immunoprecipitable 33P orthophosphate into UGT proteins and loss of activities when protein kinase C (PKC) and/or tyrosine kinase (TK) sites are mutated provide evidence UGTs require phosphorylation. Co-localization, cross-linking and co-immunoprecipitation of kinases and UGTs demonstrate the two enzyme systems functionally share compartment(s). Moreover, substrate and pH changes following mutation of PKC sites in UGT1A7 indicate phosphorylated serine residues participate in substrate selection. On the other hand 3 family 2B isozymes require Tyr (UGT2B7), Tyr and Ser/Thr (UGT2B15), or Ser/Thr (UGT2B17) phosphorylation. UGT2B7 substrate: 17-beta estradiol derived catechol-estrogens, (CEs), UGT2B15 (dihydrotestosterone) and UGT2B17 (dihydrotestosterone) metabolize endogenous genotoxic- or disease-causing hormonal agents. Our studies indicate the tyrosine kinase, SrcTK, carries out required phosphorylation of UGT2B7. This conclusion is based on: (a) loss of activity for Y236F- or Y438F-UGT2B7 mutant, but not either of three PKC-sites mutants, (b) pull-down of SrcTK and UGT2B7His with anti-His, (c) enhancement of recombinant 2B7 activity and phospho-Y438-2B7 by its co-transfection with wild-type or activated Src, but not dominant-negative Src, (d) Src-specific inhibitor, PP2, disruption of co-localization and cross-linking of 2B7 and Src, and (e) markedly increased labeling and activity of 2B7His previously expressed in Src/Yes/Fyn (SYF)-/- cells, phosphorylated in vitro with Src and 33PATP before affinity-purification and analysis compared to 33PATP alone. Consistent with the prediction that 2B7 is supported/phosphorylated by Src, treatment of 2B7 transfected COS-1 cells with AKAP12-siRNA to target the ER-based Src-scaffold abolished 70 to 100% of AKAP12, the active Src species, phospho-Y438-2B7 and 2B7 activity, but not 2B7 protein, which indicates AKAP12 assembles a Src signaling unit on ER-containing 2B7. Furthermore, we found 2B7-transfection of COS-1 cells protected against catechol-estrogen depurination, which is associated with initiation of breast cancer. Using matching controls and breast carcinomas, we observed a fully concordant pattern of high 2B7 activity, phospho-Y438-2B7 content, high kevels of the active Src species and Src activity in normal mammary gland versus dramatically reduced or not detectable parameters in breast carcinomas. The pattern was consistent with the prediction Src supports 2B7 capacity to detoxify catechol-estrogens associated with initiation of breast cancer. Dihydrotestosterone-metabolizing UGT2B15-- distributed primarily in liver, prostate and testis-- contains computer-detected S124, S172, Y99 and Y237 phosphorylation sites, and recombinant 2B15 transfected in COS-1 cells is transiently downregulated by curcumin signifying it undergoes regulated phosphorylation. 2B15 is irreversibly inhibited between 80 and 100 % by typical PKC inhibitors, calphostin-C, BIM, Go6976 and Rotlerin. In addition, 2B15 expressed in COS-1 is downregulated by Src-specific inhibitor PP2 or AKAP12-siRNA in a concentration dependent manner. PKC phosphorylation sites mutants, S172A and S124A, had 100 and 90 % loss of activity, respectively, while tyrosine-kinase sites mutants, Y237F and Y99F, were 85 and 50 % inactive, respectively. Treatment with SB203580 (JNK-specific) or DATS (MAP38 kinase-specific) inhibitor caused moderate downregulation of activity. Whereas anti-His pulled down 2B15-His and PKC-alpha, the two were moderately disrupted by pretreatment with BIM, Go6976 or Rotlerin. Results indicate 2B15 requires PKC-alpha phosphorylation at S172, but that Src is likely participating in a supportive or regulatory role at Y237 in the isozyme. 2B17--also distributed primarily in liver, prostate and testis-- has 50-fold greater activity toward dihydrotestosterone than 2B15 under in-vitro conditions, it contains S172, S422, Y99 and Y237 phosphorylation sites, and it is transiently downregulated by curcumin indicating it undergoes regulated phosphorylation. Moreover it is inhibited in a concentration-dependent manner by a range of PKC inhibitors: calphostin-C (80%), BIM (50 %), Rotlerin (60 %) and Go6976 (50%). Unlike 2B15, it is activated by Src-specific PP2 (50%) and Src-siRNA (20%). The isozyme shows minor negative effects following exposure to Src-scaffold, AKAP12-siRNA. While S172A mutant is 100% inactive;S422A (40%);Y237F (75 %) and Y99F (60 %) are less affected. Anti-His pull-down of 2B17-His trapped PKC-epsilon and its receptor, Beta-COP, suggesting 2B17 is dependent upon PKC-epsilon phosphorylation, but partially downregulated by Src phosphorylation. We have evidence an entirely different signaling system engages with Src phosphorylation at position Y237 creating a novel type of control. Before determining second messenger(s) responsible for constitutive signaling to support UGT phosphorylation, we confirmed that PKC-epsilon phosphorylates UGT1A7 by transcribing 1A7His mRNA and translating the same into protein in an in-vitro transcription-translation system. Secondly, the nascent protein was phosphorylated by PKC-epsilon using 33PgammaATP. Additionally, treatment of 1A7-transfected cells with PKC-epsilon-siRNA silenced 1A7 activity. Evidence for on-going constitutive signaling to support regulated phosphorylation of PKC-dependent UGT1A isozymes is based on: (a) reversible downregulation of 1A activities treated with both a protein-synthesis inhibitor and highly-specific PKC-epsilon translocation-antagonist peptides or siRNAs and (b) dramatic up-regulation of curcumin-inhibited 1A activities by specific PKC agonists such as DAG, PS and PMA. Additionally, catalase and herbiimycin-A inhibition of constitutive or hydrogen peroxide-activated UGTs demonstrated endogenous ROS-related oxidants behave as second messengers to maintain PKC-dependent signaling and UGT phosphorylation to support constitutive glucuronidation. In order to isolate UGT1-kinase(s) complex(es) containing signalinng element(s), ER-bound UGT was solubilized with zwitterionic-CHAPS- and sugar-containing BOG-detergents, which stabilized UGT1A7 activity as a complex composed of chaperone phospho-serine/threonine bindinng protein, 14-3-3, UGT1A7 and PKC-epsilon compared to loss of S162A-UGT1A7 activity due to its mutant 14-3-3 binding site. Also, determination of common donor-substrate, UDP-glucuronic acid, binding site(s) present in all UGTs that was begun several years ago in this laboratory by a student in the summer IRTA program using special computer software for modeling was completed. His modeling identified analogous strucuturally-solved UDP-glucose isomerase protein (ID:1XEL in the PDB) that predicted N-292, K-314, K315, and/or K-404 as potential UDP-glucuronic acid binding sites in UGT1A10. Mutational studies in conjunction with recently available affinity probe, 5N3-32P-UDP-Glucuronic acid, revealed both UGT1A10 and 1A7 have both a high and low affinity binding site by Scatchard analysis and that K-314 is the critical binding residue based on resolution of chemically-digested UGTs and their mutants separated in a discontinuous Tricine-SDS polyacrylamide gel syst